Poster Presentation 50th Lorne Proteins Conference 2025

Deep Mutational Scanning of SARS-CoV-2 Proteases. (#333)

Xinyu Wu 1 2 , Matthew Call 1 2 , Melissa Call 1 2
  1. Walter and Eliza Institite of Medical Research, Parkville, VIC, Australia
  2. Department of Medical Biology, University of Melbourne, Parkville, Victoria, Australia

Most viruses require either host or virally encoded proteases to complete their replication cycle. Protease inhibitors, in combination with other drugs are used in the clinic to treat human immunodeficiency virus and hepatitis C virus resulting in long-term control of chronic infection. Coronaviruses depend on two proteases for replication, and in the case of SARS-CoV-2, Main protease (Mpro) and Papain-Like protease (PLpro) have both been identified as viable drug targets. Nirmatrelvir (Pfizer) and Ensitrelvir (Shionogi) have already hit the market as successful Mpro inhibitors, however drug escape pathways to Nirmatrelvir, observed first in the laboratory are emerging in clinical isolates. Reliance on a single therapeutic is unlikely to be viable in the long-term as SARS-CoV-2 continues to evolve and thus development of alternative protease inhibitors and inhibitors to other viral non-structural proteins continues. While inhibitors for PLpro are not yet commercially available, several studies have reported compounds with nanomolar affinity and in vivo activity. As both Mpro and PLpro are validated and promising drug targets, we aimed to deepen our understanding of SARS-CoV-2 protease activity, via systematically substituting each amino acid residue and identifying those whose replacement alters protein function but not expression in cellular assays. There are over 6000 theoretical single-residue substitutions for each protease, and we tested almost all of the possible substitutions by deep mutational scanning for expression and activity to generate a comprehensive understanding of the sequence-function requirements for both proteases. In PLpro we reveal a network of residues whose mutations impact function, including those in active site residues, the wider substrate binding pockets, and at some surprisingly distant sites, pointing to a role for allostery1. In Mpro we again see that mutations in the substrate binding area as being required for activity and also uncover the residues involved in dimerisation (unpublished). Together this work provides novel insights into two proteases critical to SARS-CoV-2 replication and informs the development of new inhibitors to treat COVID19.

  1. Wu X, et al Nat Commun. 2024 Jul 23;15(1):6219. PMID: 39043718